1. Field of Invention
The present invention generally relates to electro-cell manipulation. More particularly, the invention concerns an electroporation apparatus and method for generating and applying an electric field to a material while controlling temperature of the process using a Peltier device for effective molecular introduction into cells and minimize damage to cellular tissue.
2. Description of Related Art
A cell has a natural resistance to the passage of molecules through its membranes into the cell cytoplasm. Scientists in the 1970s first discovered "electroporation," where electrical fields are used to create pores in cells without causing permanent damage to them. Electroporation was further developed to aid in the insertion of various molecules into cell cytoplasm by temporarily creating pores in the cells through which the molecules pass into the cell.
Electroporation has been used to implant materials into many different types of cells. Such cells, for example, include eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, and sperm. Furthermore, electroporation has been used to implant a variety of different materials, referred to herein as "implant materials," "implant molecules," "implant agents." Namely, these materials have included DNA, genes, and various chemical agents.
Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the implant agent and placed between electrodes such as parallel plates. Then, the electrodes apply an electrical field to the cell/implant mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM 600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc. In San Diego, Calif.
Known electroporation techniques for both in vitro and in vitro applications apply a brief high voltage pulse to electrodes positioned around the effectuating region. The electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the implant agent enter the cells. In known electroporation applications, this electric field comprises a single square wave pulse on the order of 1000 V/cm, of about 100 .mu.s duration. Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820, made by Genetronics, Inc.
U.S. Pat. No. 5,442,272 teaches of a quick connect suction electrode assembly for electroporation that includes a temperature regulating element. However, there is no suggestion to integrate temperature control with an electroporation control for efficient and effective implant material processing.
U.S. Pat. No. 5,185,071 teaches of a programmable electrophoresis apparatus using temperature controlling Peltier devices attached to the sides of a buffer chamber. This disclosure teaches only of electrophoresis applications.
Although known methods of electroporation may be suitable for certain applications, the electric field may actually damage the electroporated cells in some cases. For example, an uncontrolled electric field and generated heat may damage the cells by creating permanent pores in the cell walls. In extreme cases, the electric field may completely destroy the cell caused by overheating during an electroporation event.
Thus, existing electroporation systems may not be suitable for certain applications due to imprecise temperature control of implant agent materials and host cells during electroporation. Furthermore, many existing electroporation systems lack sufficient control over the parameters of the electric field pulses such as amplitude, duration, number of pulses during this process while simultaneously controlling the temperature of the implant materials.